Effects of Passovia ovata Mistletoe on Pro-Inflammatory Markers In Vitro and In Vivo

New agents that can suppress inflammatory responses are being sought, since chronic inflammation is associated with several pathologies. This work aims to elucidate phytochemicals from the hydroethanolic extract of mistletoe Passovia ovata (POH) and its anti-inflammatory potential. POH is submitted to HPLC-UV, qualitative analysis of chemical constituents, and flavonoid quantification. Cytotoxicity is evaluated in RAW 264.7 macrophages by MTT. LPS-stimulated RAW 264.7 cells are treated with POH and, after 48 h, the nitrite and cytokine levels are quantified. BALB/c mice are treated by gavage with POH and stimulated with λ-carrageenan to induce paw oedema or peritonitis. POH yield is 25% with anthraquinones, tannins, anthocyanins, anthocyanidins, flavonols, catechins and flavanones present and flavonoid content of 4.44 ± 0.157 mg QE/g dry weight. POH exhibits low cytotoxicity and significantly reduced (p < 0.01) nitrite, IL-1β, IL-6, and TNF-α quantification at 500 μg/mL. POH at 500 mg/kg prevents paw edema increase and also reduces inflammatory infiltrate and mast cells in the footpad. In the peritonitis model, POH does not influence cytokines levels or cell counts. Overall, POH demonstrates a high concentration of flavonoids and prominent effects in the reduction in pro-inflammatory markers in vitro and in the inhibition of paw oedema.


Introduction
Inflammation is a response to a harmful stimulus created by an internal or external agent of microbial, autoimmune, allergic, metabolic, and/or physical origin [1]. This response can range from being local and delimited to being a systemic inflammation, with a broad and complex sequence of events [2].
The inflammatory response involves leukocyte cells such as macrophages, neutrophils, and lymphocytes. These cells release pro-inflammatory cytokines, vasoactive amines, peptides, and acute phase proteins, which orchestrate inflammation. This process also demands a balance between neutralization of an agent and control to heal and restore the damaged area [3].
Chronic inflammation can be involved in several pathologies such as cardiovascular diseases, autoimmune and neurodegenerative diseases, and cancer [4], as pro-inflammatory cytokines, such as IL-1, IL-6, and TNF-α, are involved in the increase in platelet activity, interference of endothelial cells functions, activation of plasma coagulation cascade, reduction in fibrinolytic action, increase in platelet reactivity, and reduction in physiological anticoagulants [5]. Several problems have been associated with current anti-inflammatory therapy such as adverse effects and drug resistance. Therefore, new ways of controlling the inflammatory process are sought. In this context, medicinal plants emerge as a therapeutic alternative, and although popular use may suggest potentially important species, research is essential to prove their pharmacological effect [6].
Loranthaceae and Santalaceae form two of the largest families of mistletoes [7], a group of hemiparasitic plants that through roots named haustorium obtain water and minerals from the host plant [8]. The genus Passovia represents neotropical mistletoes from the Loranthacea family [9], a group that has been recognized for many years for its therapeutic properties [10]. Passovia ovata is a species with ovate leaves, apex usually acute or attenuated, and little-branched terminals [9]. It can be considered a generalist species, as it tends to have a wider geographic distribution. This species is a facultative aluminum accumulator plant; therefore, regardless of whether the host plant accumulates aluminum or not, P. ovata will not show symptoms of deficiency or toxicity [11].
To our knowledge, no previous study has been reported on the anti-inflammatory activity of P. ovata. Therefore, we investigated the phytochemical and immunomodulatory properties of the hydroethanolic extract from leaves of P. ovata using cultured RAW 264.7 cells and murine in vivo models.

Yield and Physicochemical Properties
The extraction yield of POH was 25%. POH was submitted for analysis by highperformance liquid chromatography photodiode array detection method (HPLC-UV) ( Figure 1).
inflammatory cytokines, such as IL-1, IL-6, and TNF-α, are involved in the increase in platelet activity, interference of endothelial cells functions, activation of plasma coagulation cascade, reduction in fibrinolytic action, increase in platelet reactivity, and reduction in physiological anticoagulants [5]. Several problems have been associated with current anti-inflammatory therapy such as adverse effects and drug resistance. Therefore, new ways of controlling the inflammatory process are sought. In this context, medicinal plants emerge as a therapeutic alternative, and although popular use may suggest potentially important species, research is essential to prove their pharmacological effect [6].
Loranthaceae and Santalaceae form two of the largest families of mistletoes [7], a group of hemiparasitic plants that through roots named haustorium obtain water and minerals from the host plant [8]. The genus Passovia represents neotropical mistletoes from the Loranthacea family [9], a group that has been recognized for many years for its therapeutic properties [10]. Passovia ovata is a species with ovate leaves, apex usually acute or attenuated, and little-branched terminals [9]. It can be considered a generalist species, as it tends to have a wider geographic distribution. This species is a facultative aluminum accumulator plant; therefore, regardless of whether the host plant accumulates aluminum or not, P. ovata will not show symptoms of deficiency or toxicity [11].
To our knowledge, no previous study has been reported on the anti-inflammatory activity of P. ovata. Therefore, we investigated the phytochemical and immunomodulatory properties of the hydroethanolic extract from leaves of P. ovata using cultured RAW 264.7 cells and murine in vivo models.

Yield and Physicochemical Properties
The extraction yield of POH was 25%. POH was submitted for analysis by high-performance liquid chromatography photodiode array detection method (HPLC-UV) (Figure 1). omatogram revealed a significant presence in the content of major constituents phenólicos with absorband the phytochemical screening results of POH (Table 1) reveal the presence of anthraquinones, tannins, anthocyanidins, flavonols, catechins, and flavanones. The total flavonoid content in POH was 4.44 /g dry weight. HPLC-UV chromatogram revealed a significant presence in the content of major constituents phenólicos with absorbance in 254 nm and the phytochemical screening results of POH (Table 1) reveal the presence of anthraquinones, tannins, anthocyanins, anthocyanidins, flavonols, catechins, and flavanones. The total flavonoid content in POH was 4.44 ± 0.157 mg QE/g dry weight. The effect of POH on the viability of RAW 264.7 macrophages ( Figure 2).  Abbreviations: sodium hydroxide (NaOH); potassium hydroxide (KOH); hydrochloric acid (HCl).

Effects of the Hydroethanolic Extract of P. ovata on Macrophages RAW 264.7
The effect of POH on the viability of RAW 264.7 macrophages ( Figure 2).

Figure 2.
Passovia ovata hydroethanolic extract in vitro toxicity in RAW 264.7 macrophages. Cell viability percentages obtained by the MTT assay after 24 h (A) and 48 h (B) from treatment with concentrations from 1000 to 1.9 μg/mL of the extract. Control group received only RPMI 1640 medium. Data represent the mean ± standard for three different experiments performed in triplicate. Abbreviation: microgram per milliliter (μg/mL).
After testing by MTT, POH demonstrated low cytotoxicity as it did not reduce cell viability in macrophages RAW 264.7 submitted to the same treatment (125, 250, and 500 μg/mL) used in the other in vitro assays. Cytotoxicity also did not increase with increasing treatment time. In LPS-stimulated RAW 264.7 macrophages, POH concentration of 500 μg/mL significatively (p < 0.01) reduced nitrite ( Figure 3A), IL-1β ( Figure 3B), IL-6 ( Figure  3C), and TNF-α ( Figure 3D). The same was observed in cells treated with dexamethasone at 5 μg/mL. The quantification of nitrite and cytokines in the group stimulated with LPS was significantly (p < 0.05) higher when compared to the control group (unstimulated and untreated). After testing by MTT, POH demonstrated low cytotoxicity as it did not reduce cell viability in macrophages RAW 264.7 submitted to the same treatment (125, 250, and 500 µg/mL) used in the other in vitro assays. Cytotoxicity also did not increase with increasing treatment time. In LPS-stimulated RAW 264.7 macrophages, POH concentration of 500 µg/mL significatively (p < 0.01) reduced nitrite ( Figure 3A), IL-1β ( Figure 3B), IL-6 ( Figure 3C), and TNF-α ( Figure 3D). The same was observed in cells treated with dexamethasone at 5 µg/mL. The quantification of nitrite and cytokines in the group stimulated with LPS was significantly (p < 0.05) higher when compared to the control group (unstimulated and untreated).

Paw Edema
Edema thickness was used to evaluate the effect of POH treatment. Edema thickness was lower than the PBS group after two, three, and four hours of treatment with dexamethasone, and after two and four hours of treatment with POH at 250 mg/kg and 500 mg/kg doses ( Figure 4). Table 2 also shows the effect of POH or standard drug dexamethasone in the reduction in paw edema in stimulated BALB/c mice after one, two, three, and four hours of treatment. The subcutaneous administration of λ-carrageenan caused an increase in footpad thickness and some treatments significantly prevented the edema: POH Results are expressed as the mean ± SD of three different experiments performed in triplicate. # p < 0.01 comparing the untreated and unstimulated group; * p < 0.05, when compared with the group stimulated with LPS, after Kruskal-Wallis test and Dunn's multiple comparison analysis. Abbreviations: lipopolysaccharide (LPS); sodium nitrite (NaNO2); interleukin-6 (IL-6); interleukin-1 beta (IL-1β); tumor necrosis factor alpha (TNF-α); micromole per milliliter (μM/mL); cells/mL (number of cells per milliliter); pg/mL (picograms per milliliter).

Paw Edema
Edema thickness was used to evaluate the effect of POH treatment. Edema thickness was lower than the PBS group after two, three, and four hours of treatment with dexamethasone, and after two and four hours of treatment with POH at 250 mg/kg and 500 mg/kg doses ( Figure 4). Table 2 also shows the effect of POH or standard drug dexamethasone in the reduction in paw edema in stimulated BALB/c mice after one, two, three, and four hours of treatment. The subcutaneous administration of λ-carrageenan caused an increase in footpad thickness and some treatments significantly prevented the edema: POH administered at a dose of 500 mg/kg (p < 0.01 and p < 0.05) or 250 mg/kg (p < 0.05) and dexamethasone at 5 mg/kg dose (p < 0.0001).

Figure 4.
Kinetic of edema thickness in millimeters of paw edema induced by 1% λ-carrageenan in BALB/c mice treated with hydroethanolic extract of Passovia ovata at 125, 250, or 500 mg/kg or dexamethasone at 5 mg/kg. Normal group is representative of untreated and unstimulated group and control is representative of the group induced with λ-carrageenan and treated with PBS. Data represent mean ± standard deviation of experiment of three different experiments performed in quintuplicate * p < 0.05; ** p < 0.01; **** p < 0.0001, after analysis of variance (two-way ANOVA) and Bonferroni's multiple comparisons test when compared with the control group induced with λ-carrageenan and treated with PBS. Abbreviations: P. ovata hydroethanolic extract (POH); dexamethasone (dexa), PBS (phosphate-buffered saline).
Footpad histological images of tissue fragments stained with hematoxylin and eosin demonstrated a greater tissue preservation in the non-stimulated group ( Figure 5A) and in groups treated with 500 mg/kg of POH ( Figure 5E) and dexamethasone ( Figure 5F) than in the untreated and non-stimulated group. A higher inflammatory infiltrate was observed in the stimulated and PBS-treated group ( Figure 5B) and in the group treated with the lowest dose (125 mg/kg) of POH ( Figure 5C).
After histological microscopic analysis of footpad tissue, it was observed that inflammatory infiltrate was present in all groups of animals used in the experiment, being discreet (+) in unstimulated and untreated group, intense (+++) in group stimulated and treated with PBS, and intense (+++), moderate (++), and discrete (+) in groups treated with 125, 250, and 500 mg/kg of POH and 5 mg/kg of dexamethasone, respectively (Table 3).  After histological microscopic analysis of footpad tissue, it was observed that inflammatory infiltrate was present in all groups of animals used in the experiment, being discreet (+) in unstimulated and untreated group, intense (+++) in group stimulated and treated with PBS, and intense (+++), moderate (++), and discrete (+) in groups treated with 125, 250, and 500 mg/kg of POH and 5 mg/kg of dexamethasone, respectively (Table 3). In mast cells from histological footpad slides stained with toluidine blue (Figure 6), the stimulated and PBS-treated group showed a significant increase (p < 0.001) in the number of mast cells when compared to the non-stimulated and untreated group. The groups treated with 500 mg/kg of POH, or dexamethasone (5 mg/kg) had a significant reduction, p < 0.001 and p < 0.0001, respectively, in mast cells, when compared to the stimulated and PBS-treated control ( Figure 6G). In mast cells from histological footpad slides stained with toluidine blue (Figure 6), the stimulated and PBS-treated group showed a significant increase (p < 0.001) in the number of mast cells when compared to the non-stimulated and untreated group. The groups treated with 500 mg/kg of POH, or dexamethasone (5 mg/kg) had a significant reduction, p < 0.001 and p < 0.0001, respectively, in mast cells, when compared to the stimulated and PBS-treated control ( Figure 6G). Data are expressed as mean ± standard deviation and images are representative of three independent experiments carried out in quintuplicate. # p < 0.001 compared with control group; *** p < 0.001, **** p < 0.0001 compared with PBS group, after analysis of variance (two-way ANOVA) followed by Kruskal-Wallis followed by Dunn's multiple. Abbreviations: lambda carrageenan (λ-carrageenan. The sign + or -represents group submitted or not to λ-carrageenan inoculation.

λ-Carrageenan-Induced Peritonitis
As shown in Figure 7A, the induction of peritonitis by carrageenan stimulated a sig- expressed as mean ± standard deviation and images are representative of three independent experiments carried out in quintuplicate. # p < 0.001 compared with control group; *** p < 0.001, **** p < 0.0001 compared with PBS group, after analysis of variance (two-way ANOVA) followed by Kruskal-Wallis followed by Dunn's multiple. Abbreviations: lambda carrageenan (λ-carrageenan. The sign + or -represents group submitted or not to λ-carrageenan inoculation.

λ-Carrageenan-Induced Peritonitis
As shown in Figure 7A, the induction of peritonitis by carrageenan stimulated a significant (p < 0.01) increase in cell migration to the peritoneal cavity when compared to the untreated group (0.957 × 10 7 cells/mL vs. 3.504 × 10 6 cells/mL). The only group that showed a significant reduction in cell count ( Figure 7A, p < 0.01), IL-1β ( Figure 7B, p < 0.05), and IL-6 ( Figure 7C, p < 0.01) quantification in serum was the one treated with dexamethasone (5 mg/kg) (0.594 × 10 7 cells/mL). The hematological evaluation consisted of red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), leukocyte counts (WBC), and platelet counts (PLT). No statistical alterations were observed between the groups, as seen in Table 4. Figure 6. Paw edema of BALB/c mice inoculated with λ-carrageenan and treated with Passovia ovata hydroethanolic extract (POH). Histological analysis by staining with toluidine blue in group (A) unstimulated and untreated group; (B) inoculated with λ-carrageenan and treated with PBS solution; (C) treated with 125 mg/kg of POH and inoculated with λ-carrageenan; (D) treated with 250 mg/kg of POH and inoculated with λ-carrageenan; (E) treated with 500 mg/kg of POH and inoculated with λ-carrageenan; (F) treated with dexamethasone (5 mg/kg) and inoculated with λ-carrageenan. (G) Quantification of mast cells in these groups. Arrows shows mast cells. Data are expressed as mean ± standard deviation and images are representative of three independent experiments carried out in quintuplicate. # p < 0.001 compared with control group; *** p < 0.001, **** p < 0.0001 compared with PBS group, after analysis of variance (two-way ANOVA) followed by Kruskal-Wallis followed by Dunn's multiple. Abbreviations: lambda carrageenan (λ-carrageenan. The sign + or -represents group submitted or not to λ-carrageenan inoculation.

Discussion
P. ovata is a neotropical mistletoe that has not been explored for its pharmacological potential, but studies reporting the promising effects of other mistletoes led us to explore its chemical compounds and immunomodulatory effect.
To better evaluate the composition, POH analysis was performed through HPLC, a widely used tool to evaluate plant origin extracts sensitively and accurately as a prerequisite for its standardization [12].
The total flavonoids content in POH was higher than the ones found in other mistletoes in similar assays such as the alcoholic extract from leaves of Phthirusa sp, (10.51 ± 1.81 mg QE/g) [18] and methanolic extract from Dendrophthoe pentandra (L.) Miq. (4.25 ± 21.21 mg EQ/g) [19]. Flavonoids are produced in plants to help fight oxidative stress and act as growth regulators. Many flavonoids isolated from plant extracts have shown anti-inflammatory effect, being, therefore, an important parameter for the elaboration of new drugs against inflammatory disorders [20].
In the in vitro assay, POH showed low cytotoxicity in RAW 264.7 macrophages after 24 and 48 h treatment, encouraging their possible future application. To evaluate the potential anti-inflammatory effect of POH, macrophages were stimulated with LPS, and cytokines and nitrite were quantified.
Macrophages form a heterogeneous cell population that can be considered classically activated by LPS and/or Th1 cytokines, which generates a pro-inflammatory profile named M1. Macrophages M1 trigger a high production of cytokines such as TNF-α, IL-12, IL-6, and IL-1β and higher amounts of nitric oxide (NO). Alternative activation is stimulated by Th2 cytokines such as IL-13 and IL-4 and defines a population with immunoregulatory and anti-inflammatory functions marked by the production of cytokines such as TGF-β and IL-10. While the M1 profile encourages the elimination of the stimulus, the M2 profile seeks to limit inflammation and promote tissue recovery and remodeling [21].
In the evaluation of potentially therapeutic compounds in macrophages, the measurement of nitrite is an important parameter as it represents an indirect way of measuring NO, a gas with signaling, regulatory, and microbicidal functions. It is produced by macrophages under circumstances that stimulate inflammation and induce them to a M1 profile [22]. After 48 h treatment with POH at 500 µg/mL, there was observed a nitrite quantification reduction in LPS-stimulated macrophages. Previous studies indicated that hydroethanolic extract from S. vulgaris mistletoe at 80 and 160 µg/mL concentrations significantly reduced the production of NO in the J774 macrophages strain [23], and the methanolic fraction from leaves of Phragmanthera capitata at 25 µg/mL concentration also reduced nitrite levels [24].
Macrophages are among the main sources of cytokines when exposed to factors that stimulate the immune response [25]. LPS is an endotoxin that induces macrophage activation, formation of self-amplifying loops, and cytokine production [26] through Tolllike receptors (TLR) [27].
In the evaluation of plant-derived compounds, IL1-β, IL-6, and TNF-α are among the main mediators used as parameters for evaluating possibly anti-inflammatory cellular responses [28]. Here, the same concentration of POH (500 µg/mL) that inhibited NO production also reduced the quantification of IL-1β, IL-6, and TNF-α. These cytokines are considered pro-inflammatory and are liberated when a stimulus, such as LPS from Gramnegative bacteria, activates TLR4 [29]. Macrophages isolated from BALB/c mice when treated with Viscum album coloratum, known as Korean mistletoe, also showed reduction on the quantification of these cytokines [30].
In the paw edema test, parameters related to the reduction in edema induced by the phlogogen agent and through the histological analysis of the inflammatory infiltrate and mast cell count of footpad tissue were evaluated. Mast cells are modulator cells of Plants 2023, 12, 1814 9 of 15 the immune system that act in acute inflammatory responses, such as those that occur in allergic reactions and in the elimination of pathogens [31]. They are among the first cells to act against antigens and initiate local inflammation mechanisms [32]. The model of inflammation induced by carrageenan involves an early phase in which mast cells release histamine and a later phase in which there is neutrophil migration and increased production and release of cytokines and prostaglandin [33].
In our work, the concentration of 500 mg/kg of POH induced the bigger reduction in paw edema, as well as a reduction in mast cells, events that may be related with the activation and release of granules and with mediators such as histamine that increase the permeability of the vessels and consequently cause the release of liquid into the extracellular environment, promoting local edema [34]. In a trial with another mistletoe, also of the Loranthacea family, S. vulgaris, the ethanol leaf extract reduced edema at 50 and 100 mg/kg doses [23].
Despite the results in the paw edema test, in the peritonitis model in BALB/c mice, treatment with POH did not demonstrate a reduction in the cell count of the intraperitoneal lavage and in cytokines IL-6 and IL-1β, although a visible trend in the reduction in cytokines is perceptible as the treatment dose increases. In these assays, only the standard drug dexamethasone showed a reduction effect in cell count and in cytokines IL-6 and IL-1β quantification.
The inflammatory process is highly complex and involves several cell types and at least 15 types of cytokines, adrenocorticotrophic hormones, and acute phase proteins, which manifest as features of redness, edema, heat, and pain [35]. Therefore, our work is a preliminary trial on the effect of this mistletoe on pro-inflammatory markers, but further elucidation of the effects of derived compounds from P. ovata on the immune system will be better determined in future trials.

Plant Material and Hydroethanolic Extract Preparation
The leaves and stems of P. ovata (Pohle x DC.) Tiegh. were collected in July 2019 at the Farm School of the State University of Maranhão-UEMA, located in the city of São Luís, Brazil, where the plant was parasitizing a mango tree (Mangifera indica). A voucher specimen was deposited with the number 5516 and identified by the Herbarium Rosa Mochel, UEMA. The genetic heritage material access was granted by the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado-SisGen) with reference record AFC60DB. P. ovata hydroethanolic extract (POH) was obtained by maceration (20% w/v) during a period of 12 days, with solvent (ethanol 70%, Êxodo Científica, Hortolândia, Brazil) changes every three days. The final extract was concentrated in a rotary evaporator (Fisatom 802, São Paulo, Brazil) and lyophilized at −70 • C (Terroni Enterprise, São Carlos, Brazil). The yield of the POH was calculated according to the following formula [36]: Total yield (%) = mass o f the extract in grams mass o f the dried plant material in grams × 100

Phytochemical Screening by Qualitative Analysis
To identify the main classes of secondary metabolites, a qualitative phytochemical analysis was performed. The tests were based on color change, precipitate formation, or foam after the addition of reagents, and resulted in the analysis of the presence of anthraquinones, saponins, tannins, anthocyanidins, chalcones, leucoanthocyanidin, catechins, flavanones, flavones, flavanols, xanthones, aurones, flavonols, and coumarins [37,38]

Flavonoid Quantification
The quantification of the flavonoid content was performed in a 96-well microplate (Jet Biofil, Guangzhou, China). In each well, 200 µL of POH (1 mg/mL) and 100 µL of 2% aluminum chloride (AlCl 3 ) (Êxodo Científica, Hortolândia, Brazil) in methanol (Êxodo Científica, Hortolândia, Brazil) were added. After incubation in the dark for 30 min, the reading was performed in a spectrophotometer (Biochrom Ltd., Cambridgeshire, UK) at 420 nm, and the obtained absorbances were interpolated from the quercetin (Sigma-Aldrich, St. Louis, MO, USA) calibration curve. All tested solutions were made in triplicate and the experiment was repeated twice. Blank samples were made with 300 µL of POH. Results were expressed in QE (quercetin equivalents) in mg per g of plant sample [39].

Cytotoxicity of the Hydroethanolic Extract of P. ovata on RAW 264.7 Macrophages
The evaluation of cytotoxicity was performed according to the adaptation of the viability assay using 3-4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) [40]. Cells were added at 2 × 10 6 /mL in 100 µL of RPMI 1640 medium (Gibco, Gainthersburg, MD, USA), supplemented with 10% FBS and incubated overnight at 37 • C with 5% CO 2 . After this period, the medium was removed, and cells were treated with concentrations of 1000 to 1.9 µg/mL of POH diluted in RPMI medium (Gibco, Gainthersburg, MD, USA). Two and a half hours before completing each treatment time (24 or 48 h), 10 µL of 5 mg/mL MTT (Sigma-Aldrich, St. Louis, MO, USA) was added with further incubation until completion of treatment. Subsequently, the cell medium was aspirated, and 100 µL of dimethyl sulfoxide (DMSO at 99.5%; Sigma-Aldrich, St. Louis, MO, USA) was added. The negative control consisted of medium-only wells, and the positive control consisted of cells and 1% DMSO. The absorbance values were acquired in a spectrophotometer (Biochrom Ltd., Cambridge, UK) with a wavelength of 570 nm, and a dose response curve was made to determinate the concentration that inhibits 50% of the cells (IC 50 ).

Paw Edema Model
The evaluation of anti-inflammatory activity was performed on a model of paw edema induced by λ-carrageenan [33,41]. Mice were separated into six groups with five animals each: (1) negative control group: animals treated with PBS by gavage without stimulation; (2) positive control group: animals treated with PBS by gavage and stimulated with λ-carrageenan; (3) standard drug group: animals treated with dexamethasone (5 mg/kg) intramuscular and stimulated with λ-carrageenan; (4) animals treated with 500 mg/kg of POH by gavage and stimulated with λ-carrageenan; (5) animals treated with 250 mg/kg of POH by gavage and stimulated with λ-carrageenan; and (6) animals treated with 125 mg/kg of POH by gavage and stimulated with λ-carrageenan. One hour after treatment, the edema was induced with 25 µL of 1% λ-carrageenan in phosphate-buffered saline (PBS) inoculated via sub-plantar tissue of the right hind paw, except for group 1, which received only 25 µL of PBS in the right hind paw. After 1, 2, 3, and 4 h from stimulation, pad swelling at both paws was measured using a Schnelltaster dial gauge caliper (Kröplin GmbH, Hessen, Germany). The animals were euthanized with ketamine (100 mg/kg, Syntec, Barueri, Brazil) and xylazine (20 mg/kg, Syntec, Barueri, Brazil) in a 1:1 ratio intraperitoneally. The difference of thickness between the inoculated pad and the non-inoculated pad was determined and the inhibition of edema in percentage was calculated.
Paw edema tissue fragments were collected for histological analysis of the inflammatory tissue. Tissue fragments were fixed for 48 h in 10% formaldehyde (Dinâmica Química Contemporânea, Indaiatuba, Brazil) solution. Then they were dehydrated, embedded in pure paraffin (Solven ® , Hortolandia, Brazil), and sectioned at 5 µm in microtome (LUPE MRP03, LupeTec, São Carlos, Brazil). Tissues were submitted to Hematoxylin (Alphatec, Rio de Janeiro, Brazil) and Eosin (Isofar, Rio de Janeiro, Brazil) staining for analysis of the inflammatory infiltrate under light microscopy (Leica Microsystems, Mannheim, Germany) at 40× magnification. Representative areas were selected in five fields and a qualitative scale was used to classify the infiltrate: (+): discreet; (++): moderate; and (+++): intense. For the quantification of mast cells in the inflammatory infiltrate, toluidine blue (Alphatec, Rio de Janeiro, Brazil) staining was used. Representative areas were selected in five fields for counting under a light microscope with an approximate 40× magnification (Leica Microsystems, Mannheim, Germany). Purple-stained cells (interpreted as mast cells) were counted.

λ-Carrageenan-Induced Peritonitis
The inflammatory peritonitis model was also performed [42]. Stimulated groups were inoculated with 250 µL of 1% λ-carrageenan intraperitoneally, or 250 µL of PBS (negative control), 1 h after treatment. The experiment consisted of six groups with five animals each, divided the same way as the paw edema model. Four hours after λ-carrageenan stimulation, mice were euthanized with a 1:1 mixture of ketamine (100 mg/kg, Syntec, São Paulo, Brazil) and xylazine (20 mg/kg, Syntec, São Paulo, Brazil) administered intramuscularly. Blood was collected through intracardiac puncture for ELISA cytokines IL-1β and IL-6 measurement from the serum. Intraperitoneal lavage with PBS was also performed for further total leukocyte count in the Neubauer chamber.

Nitrite and Cytokines Quantification
Nitric oxide (NO) evaluation was indirectly determined by nitrite concentration by the Griess colorimetric method [43]. Thus, 50 µL of supernatant from each well was added to 50 µL of Griess reagent

Statistical Analysis
Results were represented as mean ± standard deviation and analyzed by a Kruskal-Wallis test with Dunn's post-test or by two-way ANOVA with Bonferroni comparisons in the Graph Pad 9.0 software package (GraphPad Software, San Diego, CA, USA). Significant differences were considered as p < 0.05.

Conclusions
Mistletoes are well-known for their immunomodulatory potential mechanisms. Here, we developed an in vitro and in vivo study on a yet-unexplored species of neotropical mistletoe named P. ovata. The phytochemical profile showed a plant rich in secondary chemical compounds with high pharmacological potential, in addition to high quantification of flavonoids. The hydroethanolic extract from the leaves of P. ovata demonstrated low cytotoxicity and ability to reduce nitrite and cytokines considered pro-inflammatory at our highest tested dose (500 µg/mL). In vivo assays with BALB/c mice showed that the oral treatment with a higher dose of the extract (500 mg/kg) reduced mast cell migration and paw-stimulated edema, but in another in vivo inflammatory model with peritonitis in BALB/c mice, treatment with the extract did not influence intraperitoneal lavage cell counts and serum cytokine concentrations.
In general, we conclude that the hydroethanolic extract of P. ovata leaves is rich in secondary chemical compounds with a high concentration of flavonoids, has immunomodulatory effect in vitro, and can inhibit the formation of paw edema and mast cell count in the paw tissue of mice. This is the first report on the therapeutic effects of P. ovata; therefore, we encourage that, in further studies, the pathways involved in the immunomodulatory effect, as well as the use of other inflammatory models and isolation of active compounds, are performed.